Acquired resistance to drugs targeting proteins essential for cancer survival is a major hurdle for the effective treatment of cancer. In most cases the molecular components that drive the evolution and maintenance of drug resistance are not well defined, but are likely to be conserved amongst cancer subtypes. Thus, the identification of cellular pathways important for cancer drug resistance is critical for the development of new treatment strategies. A clear example of the evolution of drug resistance in a cancer population is resistance to Poly ADP-Ribose Polymerase (PARP) inhibitors (PARPi) in BRCA2-deficient cancers. Both Breast Cancer 2 susceptibility protein (BRCA2) and PARP1 function in DNA repair. Loss-of-function of either gene is not lethal, however, loss of both leads to an accumulation of double-strand breaks and cell death. This synthetic lethal interaction has made PARPi an attractive drug therapy for BRCA2-deficient cancers of the breast, prostate, and ovary; however, PARPi resistance arises from the expression of BRCA2 reversion alleles encoding a mutated, but functional, BRCA2 protein. The revertant BRCA2 protein is missing large parts of the wild-type protein and likely has increased instability and dependence on molecular chaperones. Additionally, the expression of mutated versions of BRCA2 from multiple alleles in PARPi-resistant cells likely places a significant burden on the proteostatic machinery that exists to sense and respond to changes in cellular protein folding requirements. It follows that to maintain the expression of this mutant protein and to alleviate challenges to the cells protein folding capacity, PARPi exerts a selective pressure resulting in dependence on altered proteostasis pathways for survival. I propose to test this hypothesis by addressing the following aims: 1) Characterize the dependence and effects of BRCA2 revertant protein expression on protein quality control and stress response pathways. 2) Determine whether PARPi-resistant cancer cells with BRCA2 reversion alleles rely on proteostasis pathways for survival. 3) Use an unbiased genome wide RNA interference screen to identify genes required for the survival and/or maintenance of drug resistance of PARPi-resistant cancer cells with BRCA2 reversion alleles. We expect that by the completion of these proposed studies, we will have identified the components of protein quality control and stress response pathways that are critical for the survival and/or maintenance of drug resistance in PARPi-resistant tumors that re-acquire functional BRCA2 proteins through reversion mutations. The components and pathways identified by this work will provide proof- of-concept mechanisms that may be exploited in developing new treatment strategies to prevent the escape of drug resistant populations of cancer cells. !